Stroke is a leading cause of death, long-term disability, and socioeconomic costs, highlighting the urgent need for more effective treatments. Intravenous administration of tissue plasminogen activator (tPA) is the only FDA-approved therapy to re-establish cerebral blood flow. But because of increased risk of hemorrhage beyond 4.5 hr post stroke, few stroke patients (<5%) benefit from t-PA. Further, t-PA disrupts the blood-brain barrier integrity (BBB) and is neurotoxic, aggravates reperfusion injury. Reactive oxygen species (ROS), generated soon after ischemia and during reperfusion and thereafter, are considered the main mediators of ischemia/ischemia reperfusion injury. To support this notion, in our previous studies, we showed that sequential treatment with t-PA first, followed by delivery of the antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT) encapsulated in nanoparticles (nano-SOD/CAT), both administered via carotid artery 3 hr post ischemia in a thromboembolic rat stroke model, significantly reduced reperfusion injury than did tPA alone. This sequential treatment neutralized elevated ROS levels, inhibited vascular leakage/prevented edema formation, reduced inflammation, and protected neuronal cells from apoptosis. Most important, we found out that the sequential treatment stimulated migration of neuronal and circulating progenitor cells into the infarcted brain, whereas tPA alone inhibited that movement. Based on these promising results, we designed a novel dual-action tPA nanoconjugate ? tPA conjugated to nano-SOD/CAT ? to achieve neuro and vascular protection from reperfusion injury while retaining the thrombolytic effects of tPA. Our preliminary data show that the tPA nanoconjugate a) has markedly better thrombolytic effects than tPA alone (at 1/4 of the dose of tPA, the conjugate yielded the same degree of thrombolysis), b) causes no tPA-associated neurotoxicity, and c) when given via intravenous (tail vein) injection at 6 hr post stroke, effectively reduces infarct volume, resulting in improved neurological recovery over time and increased survival (~85%), significantly more so than in rats given tPA alone (~20%). We hypothesize that our tPA nanoconjugate, with its sustained neuroprotective effects from oxidative stress and significantly better thrombolytic effects than t-PA alone, could overcome the limitations of tPA alone in minimizing reperfusion injury and achieving neurological/functional recovery, even if treatment is delayed. Ou goal is to investigate and develop our tPA nanoconjugate as a safe and effective treatment for stroke. Specific aims are: AIM 1: To analyze the neuroprotective/thrombolytic effects of the tPA nanoconjugate. AIM 2: To evaluate the extent of neurological recovery and motor functions over time. AIM 3: To confirm the brain repair mechanisms following ischemic/reperfusion injury. An effective treatment could benefit more stroke patients than can be treated with tPA alone because of the extended window of treatment and reduced risk of hemorrhagic complications. Such a therapy could significantly diminish the extent of post-stroke disability.